(a) Field of the Invention
The invention relates to novel oligonucleotide chimera used as therapeutic agents to selectively prevent gene transcription and expression in a sequence-specific manner. In particular, this invention is directed to the selective inhibition of protein biosynthesis via antisense strategy using oligonucleotides constructed from arabinonucleotide or modified arabinonucleotide residues, flanking a series of deoxyribose nucleotide residues of variable length. Particularly this invention relates to the use of antisense oligonucleotides constructed from arabinonucleotide or modified arabinonucleotide residues, flanking a series of deoxyribose nucleotide residues of variable length, to hybridize to complementary RNA such as cellular messenger RNA, viral RNA, etc. More particularly this invention relates to the use of antisense oligonucleotides constructed from arabinonucleotide or modified arabinonucleotide residues, flanking a series of deoxyribose nucleotide residues of variable length, to hybridize to and induce cleavage of (via RNaseH activation) the complementary RNA.
(b) Description of Prior Art
The Antisense Strategy
Antisense oligonucleotides (AON) are therapeutic agents that can inhibit specific gene expression in a sequence-specific manner. Many AON are currently in clinical trials for the treatment of cancer and viral diseases. For clinical utility, AON should exhibit stability against degradation by serum and cellular nucleases, show low non-specific binding to serum and cell proteins (since this binding would diminish the amount of antisense oligonucleotide available to base-pair with the target RNA), exhibit enhanced recognition of the target RNA sequence (in other words, provide increased stability of the antisense-target RNA duplex at physiological temperature), and to some extent, demonstrate cell-membrane permeability. Antisense inhibition of target gene expression is believed to occur by at least two main mechanisms. The first is “translation arrest”, in which the formation of a duplex between the antisense oligomer and its target RNA prevents the complete translation of that RNA into protein, by blocking the ability of the ribosome to recognize the complete mRNA sequence. The second, and probably more important, mechanism concerns the ability of the antisense oligonucleotide to direct the ribonuclease H (RNaseH) catalyzed degradation of the target mRNA. RNaseH is an endogenous cellular enzyme that specifically degrades RNA when it is duplexed with a complementary DNA oligonucleotide (or antisense oligonucleotide) component. For example, when an antisense DNA oligonucleotide hybridizes to a cellular mRNA via complementary base pairing, cellular RNAseH recognizes the resulting DNA/RNA hybrid duplex and then degrades the mRNA at that site. Antisense oligonucleotides that can modulate gene expression by both mechanisms are highly desirable as this increases the potential efficacy of the antisense compound in vivo.
Oligonucleotide Analogs
Oligonucleotides containing natural (ribose or deoxyribose) sugars and phosphodiester (PO) linkages are rapidly degraded by serum and intracellular nucleases, which limits their utility as effective therapeutic agents. Chemical strategies to improve nuclease stability include modification of the sugar moiety, the base moiety, and/or modification or replacement of the internucleotide phosphodiester linkage. To date, the most widely studied analogues are the phosphorothioate (PS) oligodeoxynucleotides, in which one of the non-bridging oxygen atoms in the phosphodiester backbone is replaced with a sulfur. Numerous S-DNA oligonucleotide analogues are undergoing clinical trial evaluation for the treatment of cancer, infectious diseases and other human pathologies, and some are already subjects of New Drug Application (NDA) filings. S-DNA antisense are able to elicit RNaseH degradation of the target mRNA and they are reasonably refractory to degradation by serum and cellular nucleases. However, PS-DNA antisense tend to form less thermodynamically-stable duplexes with the target RNA nucleic acid than oligodeoxynucleotides with phosphodiester (PO) linkages. Furthermore, S-DNA antisense can be less efficient at eliciting RNaseH degradation of the target RNA than the corresponding PO-DNA.
Specificity of action may be improved by developing novel oligonucleotide analogues. Current strategies to generate novel oligonucleotides are to alter the internucleotide phosphate backbone, the heterocyclic base, and the sugar ring, or a combination of these. Alteration or complete replacement of the internucleotide linkage has been the most popular approach, with over 60 types of modified phosphate backbones studied since 1994. Apart from the phosphorothioate backbone, only two others have been reported to activate RNaseH activity, i.e., the phosphorodithioate (PS2) and the boranophosphonate backbones. Because of the higher sulfur content of phosphorodithioate-linked (PS2) oligodeoxynucleotides, they appear to bind proteins tighter than the phosphorothioate (PS) oligomers, and to activate RNaseH mediated cleavage with reduced efficiency compared to the PS analogue. Boranophosphonate-linked oligodeoxynucleotides activate RNaseH mediated cleavage of RNA targets, but less well than PO- or PS-linked oligodeoxynucleotides.
Among the reported sugar-modified oligonucleotides most of them contain a five-membered ring, closely resembling the sugar of DNA (D-2-deoxyribose) and RNA (D-ribose). Example of these are α-oligodeoxynucleotide analogs, wherein the configuration of the 1′ (or anomeric) carbon has been inverted. These analogues are nuclease resistant, form stable duplexes with DNA and RNA sequences, and are capable of inhibiting β-globin mRNA translation via an RNaseH-independent antisense mechanism. Other examples are xylo-DNA, 2′-O-Me RNA and 2′-F RNA. These analogues form stable duplexes with RNA targets, however, these duplexes are not substrates for RNaseH. To overcome this limitation, mixed-backbone oligonucleotides (“MBO”) composed of either phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotide segments flanked on both sides by sugar-modified oligonucleotide segments have been synthesized (Zhao, G. et al., Biochem. Pharmacol. 1996, 51, 173; Crooke, S. T. et al. J. Pharmacol. Exp. Ther. 1996, 277, 923). Among the MBOs most studied to date is the [2′-OMe RNA]-[PS DNA]-[2′OMe RNA] chimera The PS segment in the middle of the chain serves as the RNaseH activation domain, whereas the flanking 2′-OMe RNA regions increases affinity of the MBO strand for the target RNA. MBOs have increased stability in vivo, and appear to be more effective than phosphorothioate analogues in their biological activity both in vitro and in vivo. Examples of this approach incorporating 2′-OMe and other alkoxy substituents in the flanking regions of an oligonucleotide have been demonstrated by Monia et al. by enhanced antitumor activity in vivo (Monia, P. B. et al. Nature Med. 1996, 2, 668). Several pre-clinical trials with these analogues are ongoing.
The synthesis of oligonucleotides containing hexopyranoses instead of pentofuranose sugars has also been reported. A few of these analogues have increased enzymatic stability but generally suffer from a reduced duplex forming capability with the target sequence. A notable exception is 6′→4′ linked oligomers constructed from 1,5-anhydrohexitol units which, due to their highly pre-organized sugar structure, form very stable complexes with RNA. However, none of these hexopyranose oligonucleotide analogues have been shown to elicit RNaseH activity. Recently, oligonucleotides containing completely altered backbones have been synthesized. Notable examples are the peptide nucleic acids (“PNA”) with an acyclic backbone. These compounds have exceptional hybridization properties, and stability towards nucleases and proteases. However, efforts to use PNA oligomers as antisense constructs have been hampered by poor water solubility, self-aggregation properties, poor cellular uptake, and inability to activate RNaseH. Very recently, PNA-[PS-DNA]-PNA chimeras have been designed to maintain RNaseH mediated cleavage via the PS-DNA portion of the chimera.
Arabinonucleosides and Arabinonucleic Acids (ANA)
Arabinonucleosides are isomers of ribonucleosides, differing only in the stereochemistry at the 2′-position of the sugar ring. We have previously shown that antisense oligonucleotides constructed entirely from nucleotides comprising arabinose or modified arabinose (especially 2′-F arabinose) sugars are able to elicit RNaseH degradation of the complementary target RNA (Damha, M. J. et al. JACS 1998, 120, 12976; Noronha, A. M. et al. Biochemistry 2000, 39, 7050). We also noted that the thermal stability of duplexes consisting of an arabinose oligonucleotide with RNA was less than that of the analogous DNA/RNA duplex (Noronha, A. M. et al. Biochemistry 2000, 39, 7050). In contrast however, the thermal stability of duplexes consisting of an oligonucleotide synthesized with 2′-F arabinose nucleotides hybridized with RNA is generally greater than that of the analogous DNA/RNA duplex (Damha, M. J. et al. JACS 1998, 120, 12976). Giannaris and Damha found that replacement of the phosphodiester (PO) linkage in ANA oligonucleotides with phosphorothioate (PS) linkages significantly decreased the stability of the PS-ANA/PO-RNA duplex (Giannaris, P. A.; Damha, M. J. Can. J. Chem. 1994, 72, 909). This destabilization was greater than that observed when the PO linkages of an analogous DNA oligonucleotide were replaced with S internucleotide linkages (Giannaris, P. A.; Damha, M. J. Can. J. Chem. 1994, 72, 909).
Watanabe and co-workers incorporated 2′-deoxy-2′-fluoro-□-D-arabinofuranosylpyrimidine nucleosides (2′-F-ara-N, where N═C, U and T) at several positions within an oligonucleotide primarily comprised of a PO-DNA chain and evaluated the hybridization properties of such (2′-F)ANA-DNA “chimeras” towards complementary DNA (Kois, P. et al. Nucleosides & Nucleotides 1993, 12, 1093). Substitutions with 2′-F-araU and 2′-F-araC destabilized duplex stability compared to the all-DNA/RNA duplex, whereas substitutions with 2′-F-araT stabilized the duplex. Marquez and co-workers recently evaluated the self-association of a DNA strand in which two internal thymidines were replaced by 2′-F-araT's (Ikeda et al. Nucleic Acids Res. 1998, 26, 2237). They confirmed the findings of Watanabe and co-workers that internal 2′-F-araT residues stabilize significantly the DNA double helix. The association of these (2′-F)ANA-DNA “chimeras” with complementary RNA (the typical antisense target) was not reported.
Elicitation of Cellular RNaseH Degradation of Target RNA by Antisense Oligonucleotides
One of the most important mechanisms for antisense oligonucleotide directed inhibition of gene expression is the ability of these antisense oligonucleotides to form a structure, when duplexed with the target RNA, that can be recognized by cellular RNaseH. This enables the RNaseH-mediated degradation of the RNA target, within the region of the antisense oligonucleotide-RNA base-paired duplex (Monia et al. J. Biol. Chem. 1993, 268, 14514).
RNase H selectively degrades the RNA strand of a DNA/RNA heteroduplex. RNaseH1 from the bacterium Escherichia coli is the most readily available and the best characterized enzyme. Studies with eukaryotic cell extracts containing RNase H suggest that both prokaryotic and eukaryotic enzymes exhibit similar RNA-cleavage properties, although the bacterial enzyme is better able to cleave duplexes of small length (Monia et al. J. Biol. Chem. 1993, 268, 14514). E. coli RNaseH1 is thought to bind in the minor groove of the DNA/RNA double helix and to cleave the RNA by both endonuclease and processive 3′-to-5′ exonuclease activities. The efficiency of RNase H degradation displays minimal sequence dependence and is quite sensitive to chemical changes in the antisense oligonucleotide. For example, while RNaseH readily degrades RNA in S-DNA/RNA duplexes, it cannot do so in duplexes comprising methylphosphonate-DNA, α-DNA, or 2′-OMe RNA antisense oligonucleotides with RNA. Furthermore, while E. coli RNaseH binds to RNA/RNA duplexes, it cannot cleave either RNA strand, despite the fact that the global helical conformation of RNA/RNA duplexes is similar to that of DNA/RNA substrate duplexes (“A”-form helices). These results suggest that local structural differences between DNA/RNA (substrate) and RNA/RNA (substrate) duplexes contribute to substrate discrimination.
Arabinonucleic Acids as Activators of RNaseH Activity
An essential requirement in the antisense approach is that an oligonucleotide or its analogue recognize and bind tightly to its complementary target RNA. The ability of the resulting antisense oligonucleotide/RNA duplex to serve as a substrate of RNaseH is likely to have therapeutic value by enhancing the antisense effect relative to antisense oligonucleotides that are unable to activate this enzyme. Apart from PS-DNA (phosphorothioates), PS2-DNA (phosphorodithioates), boranophosphonate-linked DNA, and MBO oligos containing an internal PS-DNA segment, the only examples of fully modified oligonucleotides that elicit RNaseH activity are those constructed from arabinonucleotide (ANA) or modified arabinonucleotide residues (International Application published under No. WO 99/67378; Damha, M. J. et al. JACS 1998, 120, 12976; Noronha, A. M. et al. Biochemistry 2000, 39, 7050). These ANA oligonucleotides retain the natural β-D-furanose configuration and mimic the conformation of DNA strands (e.g., with sugars puckered in the C2′-endo conformation). The latter requirement stems from the fact that the antisense strand of natural substrates is DNA, and as indicated above, its primary structure (and/or conformation) appears to be essential for RNaseH/substrate cleavage; the DNA sugars of DNA/RNA hybrids adopt primarily the C2′-endo conformation. ANA is a stereoisomer of RNA differing only in the stereochemistry at the 2′-position of the sugar ring. ANA/RNA duplexes adopt a helical structure that is very similar to that of DNA/RNA substrates (“A”-form), as shown by similar circular dichroism spectra of these complexes (Damha, M. J. et al. JACS 1998, 120, 12976; Noronha, A. M. et al. Biochemistry 2000, 39, 7050).
Mixed-backbone or “Gapmer” Oligonucleotide Constructs as Antisense Oligonucleotides
Mixed-backbone oligonucleotides (MBO) composed of a phosphodiester or phosphorothioate oligodeoxynucleotide “gap” segment flanked at both the 5′- and 3′-ends by sugar-modified oligonucleotide “wing” segments have been synthesized (Zhao, G. et al., Biochem. Pharmacol. 1996, 51, 173; Crooke, S. T. et al. J. Pharmcol. Exp. Ther. 1996, 277, 923). Probably the most studied MBO to date is the [2′-OMe RNA]-[PS DNA]-[2′OMe RNA] chimera. Oligonucleotides comprised of 2′-OMe RNA alone bind with very high affinity to target RNA, but are unable to elicit RNaseH degradation of that target RNA. In [2′-OMe RNA]-[PS DNA]-[2′OMe RNA] chimera oligonucleotides, the PS-DNA segment in the middle of the chain serves to elicit RNaseH degradation of the target, whereas the flanking 2′-OMe RNA “wing” regions increase the affinity of the MBO strand for the target RNA. MBOs have increased stability in vivo, and appear to be more effective than same-sequence PS-DNA analogues in their biological activity both in vitro and in vivo. Examples of this approach incorporating 2′-OMe and other alkoxy substituents in the flanking regions of an oligonucleotide have been demonstrated by Monia et al. by enhanced antitumor activity in vivo (Monia, P. B. et al. Nature Med. 1996, 2, 668). Several pre-clinical trials with these analogues are ongoing.
Nonetheless, because 2′-OMe RNA cannot elicit RNaseH activity, the DNA gap size of the [2′-OMe RNA]-[PS DNA]-[2′OMe RNA] chimera oligonucleotides must be carefully defined While E. coli RNaseH can recognize and use 2′-OMe RNA MBO with DNA gaps as small as 4 DNA nucleotides (Shen, L. X. et al 1998 Biorg. Med. Chem. 6, 1695), the eukaryotic RNaseH (such as human RNaseH) requires substantially larger DNA gaps (7 DNA nucleotides or more) for optimal degradation activity (Monia, B. P. et al 1993 J. Biol. Chem. 268, 14514). In general, with [2′-OMe RNA]-[PS DNA]-[2′OMe RNA] chimera oligonucleotides, eukaryotic RNaseH-mediated target RNA cleavage efficiency decreases with decreasing DNA gap length, and becomes increasingly negligible with DNA gap sizes of less than 6 DNA nucleotides. Thus, antisense activity of [2′-OMe RNA]-[PS DNA]-[2′OMe RNA] chimera oligonucleotides is highly dependent on DNA gap size (Monia, B. P. et al 1993 J. Biol. Chem. 268, 14514; Agrawal, S. and Kandimalia, E. R 2000 Mol. Med. Today, 6, 72).
Recently, oligonucleotides containing completely altered backbones have been synthesized. Notable examples are the peptide nucleic acids (“PNA”) with an acyclic backbone. These compounds have exceptional hybridization properties, and stability towards nucleases and proteases. However, efforts to use PNA oligomers as antisense constructs have been hampered by poor water solubility, self-aggregation properties, poor cellular uptake, and inability to activate RNaseH. Very recently, PNA-[PS-DNA]-PNA chimeras have been designed to maintain RNaseH mediated cleavage via the PS-DNA portion of the chimera
It would be highly desirable to be provided with oligonucleotides constructed from arabinonucleotide or modified arabinonucleotide residues, flanking a series of deoxyribose nucleotide residues of variable length, for the sequence specific inhibition of gene expression via association to (and RNaseH mediated cleavage of) complementary messenger RNA.